During endochondral bone formation, osteogenesis and chondrogenesis are linked and highly coordinated. However, the regulatory mechanisms responsible for coordinating osteogenesis and chondrogenesis remain unclear. Genetic mouse models have shown that the Hedgehog (Hh) pathway, which controls target genes in chondrogenesis such as PTHrP and osteogenesis such as BMP2, is essential for normal endochondral bone formation. However, despite its obvious importance, little is known of the regulatory mechanisms that are responsible for controlling Hedgehog signaling. This proposal is directed at improving understanding of the transcriptional control of the Hedgehog pathway, and specifically of Ihh, during chondrogenesis and how it may be linked to osteogenesis. Since chondrocyte differentiation occurs also at early stages of cartilage degeneration as well as during bone regeneration, this study is directly applicable to diseases such as osteoarthritis and fractures. We hypothesize that the transcription factor ATF4 is a major regulator of Hedgehog signaling during chondrogenesis, and possibly during osteogenesis as well. Further, we hypothesize that ATF4 is a direct regulator of Ihh transcription. We base this hypothesis on the following rationale. 1. We have found that ATF4 is essential for osteoblast differentiation and bone formation (Yang, et al., 2004). 2. Atf4-deficient (Atf4-/-) mice are dwarfs, suggesting a defect in growth plate chondrocytes. 3. Atf4-/- growth plate chondrocytes show several abnormalities including a delay in hypertrophic mineralization, an expansion of the hypertrophic zone, and most notably a decrease in Ihh gene expression and proliferation. These defects are reminiscent of the Ihh-/-, PPR-/-(receptor for parathyroid hormone, PTH, and PTH related protein, PTHrP), and PTHrP-/- limb phenotypes. 4. ATF4 transactivates Ihh through directly binding to a cis element of the Ihh promoter. 5. Activation of Ihh signaling restores the size of in Atf4-/- limb in an organ culture system. To test our hypothesis that ATF4 is a regulatory factor for Hedgehog signaling in chondrogenesis, we plan the following specific aims. 1. To determine the molecular mechanisms whereby ATF4 regulates Ihh transcription. 2. To address the role of ATF4 in regulating growth plate chondrocytes proliferation and differentiation in vivo. 3. To demonstrate that Ihh is a direct transcriptional target of ATF4 in vivo. PUBLIC HEALTH RELEVANCE. Indian hedgehog (Ihh) is a well-studied morphogen that regulates the coordination between chondrogenesis and osteogenesis, two processes during skeletal development and bone fracture repair. Surprisingly, investigations on the regulation of Ihh itself are lacking, particularly at the transcriptional level. In this proposal, we intend to investigate this important biological question through systematic molecular and genetic studies.